Temporal expression of the transgenic human protamine gene cluster Kathy S Stewart, M.D., Jeffrey A Kramer, Ph.D., Mark I Evans, M.D., Stephen A Krawetz, Ph.D.  Fertility and Sterility  Volume 71, Issue 4, Pages 739-745 (April 1999) DOI: 10.1016/S0015-0282(98)00548-2

FIGURE 1 Conservation of gonadal development and morphologic structure of transgenic mice of various ages. (A), Twelve-day-old mouse testis. Original magnification, ×200. (B), Fourteen-day-old mouse testis. Original magnification, ×300. (C), Twenty-one–day-old mouse testis. Original magnification, ×250. (D), Six-week-old mouse testis. Original magnification, ×200. Open arrows indicate spermatogonia, solid arrows indicate pachytene spermatocytes, open arrowheads indicate round spermatids, and solid arrowheads indicate mature spermatozoon. Fertility and Sterility 1999 71, 739-745DOI: (10.1016/S0015-0282(98)00548-2)

FIGURE 2 Comparison of testicular development between mature, age-matched transgenic and nontransgenic animals. Cellular morphology and testicular development are conserved. (A), Mature, 6-month-old transgenic mouse. Original magnification, ×200. (B), Age-matched, 6-month-old nontransgenic mouse. Original magnification, ×200. Solid arrows indicate pachytene spermatocytes, open arrowheads indicate round spermatids, and solid arrowheads indicate mature spermatozoon. Fertility and Sterility 1999 71, 739-745DOI: (10.1016/S0015-0282(98)00548-2)

FIGURE 3 Orientation of the integrated transgene. Genomic DNA was isolated from the livers of transgenic mice from several independent lines, digested to completion with BamHI, and resolved by field inversion gel electrophoresis on a 0.75% agarose gel. Cosmid clone hP3.1 digested with BamHI and lambda phage DNA digested with HindIII provided molecular weight markers as indicated in kilobases. The nucleic acids were transferred to positively charged nylon membranes and then hybridized with radiolabeled subclones 8e4 and 5f1 (data not shown). The membranes were washed at different levels of stringency to remove background hybridization and then autoradiographed at −70°C. A map of the approximately 40-kilobase construct (derived from hP3.1) used in the creation of the transgenic animals is indicated at the bottom. The genes are depicted as arrows, indicating their direction of transcription; BamHI restriction sites are shown as barbells; and the positions of the subclones used as probes are indicated by black boxes. kbp = kilobase pair. Fertility and Sterility 1999 71, 739-745DOI: (10.1016/S0015-0282(98)00548-2)

FIGURE 4 Temporal analysis of human transgene expression in mouse testes. Total RNA was isolated from testis of the nine copy homozygous line 892 (nine copy) mice at 12, 17, 28, and 80 days of age, resolved on 1.5% agarose–formaldehyde gel, and transferred to nylon membranes. Membranes were hybridized using probes specific to human PRM1, PRM2, TNP2, and mouse β-actin. The membranes were washed at different levels of stringency to remove nonspecific background hybridization and then autoradiographed at −70°C. The age of the mice used for testicular RNA isolation is indicated above each lane and the size of each message in nucleotides (nt) or kilobases (kb) is indicated along the side. Note that the β-actin probe also identifies the round spermatid–expressed smooth muscle γ-actin of approximately 1.5 kb. Fertility and Sterility 1999 71, 739-745DOI: (10.1016/S0015-0282(98)00548-2)

FIGURE 5 Schematic representation of the possible orientation of the inserted transgenes. Representations of the two most common orientations of the inserted transgene are shown. Three copies of the gene cluster are indicated in either a head-to-tail (top) or a head-to-head/tail-to-tail (bottom) orientation. The arrowheads correspond to the 3′ tail end of the transgene. The genes are indicated by lines with filled circles beneath the map and the terminal BamHI restriction sites are indicated by lines with open circles above the map. Top: Both subclone probes 8e4 and 5f1 annealed to an identical approximately 20-kilobase BamHI restriction fragment in a head-to-tail orientation. Bottom: In a head-to-head orientation, 8e4 recognized two sites in an approximately 26-kilobase BamHI restriction fragment, whereas 5f1 annealed to sites within a separate approximately 14-kilobase fragment. Fertility and Sterility 1999 71, 739-745DOI: (10.1016/S0015-0282(98)00548-2)